In the last 25 years, conjugate vaccines, comprising bacterial capsular polysaccharides (CPS) conjugated to protein carriers have been developed. Capsular polysaccharides are important immunogens found on the surface of bacteria involved in various bacterial diseases. This feature has led to them being an important component in the design of vaccines. As saccharides are T-independent antigens, generally CPS are poorly immunogenic. Conjugation to a carrier can convert T-independent antigens into T-dependent antigens, thereby enhancing memory responses and allowing protective immunity to develop.
Therefore, the most effective saccharide vaccines are based on glycoconjugates. Examples include, amongst others, the Haemophilus influenzae type b (Hib) conjugate vaccine, conjugate vaccines against Streptococcus pneumoniae and serogroup C Neisseria meningitidis (MenC). Another bacterium for which conjugate vaccines have been described is Streptococcus agalactiae, also known as ‘Group B streptococcus’, or simply ‘GBS’. The ‘B’ in ‘GBS’ refers to the Lancefield classification which is based on the antigenicity of a carbohydrate which is soluble in dilute acid and called the C carbohydrate. Lancefield identified 13 types of C carbohydrate (designated A to O) that could be serologically differentiated. The organisms that most commonly infect humans are found in groups A, B, D, and G. Within group B, strains of Streptococcus agalactiae are divided into 10 serotypes (Ia, Ib, II, III, IV, V, VI, VII, VIII and IX) based on the structure of their polysaccharide capsule.
Group B Streptococcus agalactiae causes serious disease, bacteremia and meningitis, in immunocompromised individuals and in neonates. There are two main types of neonatal GBS infection. Early onset disease occurs within 5 days of birth and is manifested by bacteremia (sepsis or infection of the blood) and pneumonia (an infection of the lungs). Late onset disease occurs from the first week of birth up to around three months after birth. Late onset disease is commonly characterized by meningitis (infection of the fluid and lining around the brain) although bacteremia and pneumonia may also occur. GBS colonises the vagina of about 25 percent of young women and is contracted vertically as a baby passes through the birth canal. Approximately 1 percent of infants born via a vaginal birth to colonised mothers will become infected with a mortality rate of between 50-70 percent.
Investigations have been conducted into the development of protein-based and polysaccharide-based vaccines against GBS. Conjugates of each of the capsular polysaccharides vaccines from GBS serotypes Ia, Ib, II, III, and V have been shown to be safe and immunogenic in humans. For example, vaccination of pregnant women with type III CPS has been demonstrated to reduce the incidence of the late onset meningitis—infants acquire protective antibodies via placental transfer and are passively immunised.
Large-scale production of capsular polysaccharide vaccines requires adequate supplies of purified capsular polysaccharides. Methods for isolating capsular polysaccharides from bacterial cells exist in the art. For example: EP0038265 discloses a method of preparing antigenic polysaccharides which comprises phenolizing the fermentation broth to lyse bacteria and release polysaccharide into the fermentation broth. EP0302887 discloses extraction of GBS type III polysaccharide by the general technique of Jennings et al. (Canadian J. Biochem. 58:112-120 (1980)).
EP1664319 describes a method for producing polysaccharide which comprises: a) using a cationic detergent to precipitate the polysaccharide or part of the contaminants from the supernatant to obtain a first polysaccharide fraction; b) using alcohol to precipitate the polysaccharide from the first polysaccharide fraction to obtain a second polysaccharide fraction; c) subjecting the second polysaccharide fraction to an alcohol precipitation in the presence of an anionic detergent, whereby the alcohol is present in a concentration which is below the concentration at which the polysaccharide precipitates; d) precipitating the polysaccharide from the soluble fraction using alcohol to obtain a polysaccharide precipitate; e) dissolving the polysaccharide precipitate and subjecting it to concentration and diafiltration.
EP1828230 describes a process for heterologous expression and secretion of complex polysaccharides in non-pathogenic, non-invasive Gram-positive bacteria.
EP1951887 and EP2004223 relate to novel strains of Staphylococcus aureus that produce type 5 capsular polysaccharide at greater levels than wild-type bacteria.
EP1051506 discloses a method for purifying capsular polysaccharides from cellular components of bacteria and culture supernatants. The method utilises alkaline treatment to lyse bacteria but this also causes hydrolysis of the base labile bond that connects the capsular polysaccharide to cellular components it also deacetylates N-acetyl groups.
However, the above methods require many steps of purification, made more complex by attachment of capsular polysaccharide to the cell wall. It is therefore the object of the invention to provide improved methods for producing capsular polysaccharides without the need for bacterial lysis or enzymatic treatment to release polysaccharide thereby simplifying purification and increasing yield.